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1.
J Sci Food Agric ; 100(3): 1195-1203, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31721226

RESUMO

BACKGROUND: The present study investigated the effect of two-step heat treatments on the structure of grass carp myofibrillar proteins (MPs) and their binding ability for selected aldehydes (hexanal, heptanal, octanal and nonanal). RESULTS: Within 30 min of the first heating step at 40 °C and 5-10 min of the second heating step at 90 °C, the enhancement of the flavor-binding ability was likely explained by the increases in surface hydrophobicity and total sulfhydryl content due to the unfolding of secondary structures of MPs through exposure of hydrophobic amino acids and sulfhydryl groups. Nevertheless, lengthy heating at 90 °C accelerated the aggregation of unfolded MPs and reduced the hydrophobic bonding sites, thus weakening the hydrophobic interactions and decreasing the resultant binding ability of MPs with aldehydes. CONCLUSION: The binding ability of aldehydes to MPs was found to be strongly influenced by changes in protein structure and surface during the two-step heating process. The results provided insight into improving the flavor characteristics of freshwater fish surimi products. © 2019 Society of Chemical Industry.


Assuntos
Aldeídos/análise , Produtos Pesqueiros/análise , Proteínas de Peixes/química , Miofibrilas/química , Animais , Carpas , Aromatizantes/química , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína
2.
J Sci Food Agric ; 100(1): 315-324, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525262

RESUMO

BACKGROUND: In order to utilize tilapia skin gelatin hydrolysate protein, which is normally discarded as industrial waste in the process of fish manufacture, we study the in vivo and in vitro angiotensin-I-converting enzyme (ACE) inhibitory activity of the peptide Leu-Ser-Gly-Tyr-Gly-Pro (LSGYGP). The aim was to provide a pharmacological basis of the development of minimal side effects of ACE inhibitors by comparative analysis with captopril in molecular docking. RESULTS: This peptide from protein-rich wastes showed excellent ACE inhibitory activity (IC50  = 2.577 µmol L-1 ) and exhibited a mixed noncompetitive inhibitory pattern with Lineweaver-Burk plots. Furthermore, LSGYGP and captopril groups both showed significant decreases in blood pressure after 6 h and maintained good digestive stability over 4 h. Molecular bond interactions differentiate competitive captopril upon hydrogen bond interactions and Zn(II) interaction. The C-terminal Pro generates three interactions (hydrogen bonds, hydrophilic interactions and Van der Waals interactions) in the peptide and effectively interacts with the S1 and S2 pockets of ACE. CONCLUSION: LSGYGP, with an IC50 value of 2.577 µmol L-1 , has an antihypertensive effect in spontaneously hypertensive rats. Through comparison with captopril, this study revealed that LSGYGP may be a potential food-derived ACE inhibitory peptide and could act as a functional food ingredient to prevent hypertension. © 2019 Society of Chemical Industry.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Captopril/química , Hipertensão/tratamento farmacológico , Peptídeos/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Captopril/administração & dosagem , Ciclídeos , Digestão , Proteínas de Peixes/química , Trato Gastrointestinal/metabolismo , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Cinética , Masculino , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Ratos , Ratos Endogâmicos SHR
3.
Dev Genes Evol ; 229(5-6): 161-181, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31486889

RESUMO

KIF3A and KIF3B are homologous motor subunits of the Kinesin II protein family. KIF3A, KIF3B, and KAP3 form a heterotrimeric complex and play a significant role in spermatogenesis. Here, we first cloned full-length kif3a/3b cDNAs from Larimichthys polyactis. Lp-kif3a/3b are highly related to their homologs in other animals. The proteins are composed of three domains, an N-terminal head domain, a central stalk domain, and a C-terminus tail domain. Lp-kif3a/3b mRNAs were found to be ubiquitously expressed in the examined tissues, with high expression in the testis. Fluorescence in situ hybridization (FISH) was used to analyze the expression of Lp-kif3a/3b mRNAs during spermiogenesis. The results showed that Lp-kif3a/3b mRNAs had similar expression pattern and were continuously expressed during spermiogenesis. From middle spermatid to mature sperm, Lp-kif3a/3b mRNAs gradually localized to the side of the spermatid where the midpiece and tail form. In addition, we used immunofluorescence (IF) to observe that Lp-KIF3A protein co-localizes with tubulin during spermiogenesis. In early spermatid, Lp-KIF3A protein and microtubule signals were randomly distributed in the cytoplasm. In middle spermatid, however, the protein was detected primarily around the nucleus. In late spermatid, the protein migrated primarily to one side of the nucleus where the tail forms. In mature sperm, Lp-KIF3A and microtubules accumulated in the midpiece. Moreover, Lp-KIF3A co-localized with the mitochondria. In mature sperm, Lp-KIF3A and mitochondria were present in the midpiece. Therefore, Lp-KIF3A/KIF3B may be involved in spermiogenesis in L. polyactis, particularly during nuclear reshaping and tail formation.


Assuntos
Proteínas de Peixes/metabolismo , Peixes/fisiologia , Cinesina/metabolismo , Espermatogênese , Espermatozoides/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Humanos , Cinesina/química , Cinesina/genética , Masculino , Filogenia , Alinhamento de Sequência , Espermatozoides/metabolismo
4.
Genes (Basel) ; 10(9)2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491991

RESUMO

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.


Assuntos
Evolução Molecular , Peixes/genética , Duplicação Gênica , Processos de Determinação Sexual , Cromossomo Y/genética , Aclimatação , Animais , Sequência Conservada , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixes/crescimento & desenvolvimento , Mutação INDEL , Masculino
5.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 8): 537-542, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397324

RESUMO

The crystal structure of haemoglobin from Atlantic cod has been solved to 2.54 Šresolution. The structure consists of two tetramers in the crystallographic asymmetric unit. The structure of haemoglobin obtained from one individual cod suggests polymorphism in the tetrameric assembly.


Assuntos
Cristalografia por Raios X/métodos , Proteínas de Peixes/química , Gadus morhua , Hemoglobinas/química , Animais , Proteínas de Peixes/metabolismo , Gadus morhua/metabolismo , Modelos Moleculares , Conformação Proteica
6.
Fish Shellfish Immunol ; 93: 567-574, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394161

RESUMO

HMGB2, a member of the high mobility group box family, plays an important role in host immune responses. However, the mechanism of action of HMGB2 is not well understood. Herein, a homologue from yellow catfish (Pelteobagrus fulvidraco) was cloned and named PfHMGB2. The deduced amino acid sequence of PfHMGB2 possessed a typical tripartite structure (two DNA binding boxes and an acid tail) and shared 90% identity with the predicted HMGB2 from I. punctatus. The mRNA of PfHMGB2 was widely distributed in all 11 tested tissues in healthy fish bodies and was significantly induced in the liver and head kidney when yellow catfish were injected with inactivated Aeromonas hydrophila. Consistently, PfHMGB2 mRNA could also be induced in yellow catfish peripheral blood leucocytes (PBL) by lipopolysaccharide. The recombinant PfHMGB2 protein was purified from E. coli BL21 (DE3):pET-28a/PfHMGB2 and showed DNA-binding affinity. Moreover, rPfHMGB2 improved the phagocytosis and proliferation activity and upregulated the mRNA expression of the pro-inflammatory cytokine TNFα in yellow catfish PBL. These results indicated that PfHMGB2 could protect yellow catfish from pathogen infection by activating PBL.


Assuntos
Peixes-Gato/genética , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteína HMGB2/genética , Proteína HMGB2/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteína HMGB2/química , Leucócitos/imunologia , Fagocitose/imunologia , Filogenia , Alinhamento de Sequência/veterinária
7.
Mar Drugs ; 17(8)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394862

RESUMO

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5-29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.


Assuntos
Colágeno Tipo I/química , Colágenos Associados a Fibrilas/química , Proteínas de Peixes/química , Pele/química , Takifugu/metabolismo , Tetraodontiformes/metabolismo , Ácidos/química , Aminoácidos/química , Animais , Ligações de Hidrogênio , Pepsina A/química , Rios , Solubilidade , Temperatura Ambiente
8.
Fish Shellfish Immunol ; 93: 406-415, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31369857

RESUMO

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia , Poli I-C/farmacologia , Polidesoxirribonucleotídeos/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Acetato de Tetradecanoilforbol/farmacologia , Proteína 1 de Ligação a Y-Box/química
9.
Phys Chem Chem Phys ; 21(35): 19298-19310, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31451813

RESUMO

The ice/water interface recognition mechanism of antifreeze proteins (AFPs) is highly contentious. Conventionally, protein adsorption on a solid surface is primarily driven by the polar interactions between the hydrophilic residues of the protein and interfacial water of the solid surface. Ice surface recognition by a type III AFP is surprising in this context where the ice binding surface (IBS) is hydrophobic. The present study provides molecular insight into the unusual interface recognition phenomenon of a type III AFP (QAE isoform) from Macrozoarces americanus. Potential of mean force calculations show that the type III AFP adsorbs on the ice surface mediated through a layer of ordered water. Molecular dynamics simulations at lower than ambient temperature reveal that the flat hydrophobic IBS induces ordering of water. The excellent geometrical synergy between the hydration water structure around the IBS and water arrangements on the pyramidal surface favours adsorption on the pyramidal plane. Mutations that interrupt the hydration shell water ordering essentially lead to less efficient adsorption, which greatly reduces the anti-freezing activity of the AFP. Binding free energy calculations of the wild-type and several mutant AFPs reveal that the binding affinity is linearly correlated with the experimentally observed thermal hysteresis activity. Therefore, binding to a specific ice plane with considerable affinity is the dictating factor of the anti-freeze activity for a type III AFP. Mechanistic insights into the ice binding process of the wild-type and different mutant AFPs obtained from this study pave the way for rational design of type III variants with much improved activity, which possesses ample industrial applicability, particularly in cryo-preservation.


Assuntos
Proteínas Anticongelantes/química , Proteínas de Peixes/química , Gelo , Perciformes , Água/química , Animais , Proteínas Anticongelantes/genética , Temperatura Baixa , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica
10.
Fish Shellfish Immunol ; 93: 702-710, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421242

RESUMO

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process used to maintain cell survival and homeostasis. A series of autophagy-related genes (Atgs) are involved in the autophagic pathway. In mammals, a growing number of studies have attributed functions to some Atgs that are distinct from their classical role in autophagosome biogenesis, such as resistance to pathogens. However, little is known about the functions of fish Atgs. In this study, we cloned and characterized an atg12 homolog from orange spotted grouper (Epinephelus coioides) (Ecatg12). Ecatg12 encodes a 117 amino acid protein that shares 94.0% and 76.8% identity with gourami (Anabas_testudineus) and humans (Homo sapiens), respectively. The transcription level of Ecatg12 was lower in cells infected with Singapore grouper iridovirus (SGIV) than in non-infected cells. Fluorescence microscopy revealed that EcAtg12 localized in the cytoplasm and nucleus in grouper spleen cells. Overexpression of EcAtg12 significantly increased the replication of SGIV, as evidenced by increased severity of the cytopathic effect, transcription levels of viral genes, levels of viral proteins, and progeny virus yield. Further studies showed that EcAtg12 overexpression decreased the expression levels of interferon (IFN) related molecules and pro-inflammatory factors and inhibited the promoter activity of IFN-3, interferon-stimulated response element, and nuclear factor-κB. Together, these results demonstrate that EcAtg12 plays crucial roles in SGIV replication by downregulating antiviral immune responses.


Assuntos
Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteína 12 Relacionada à Autofagia/química , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
11.
Fish Shellfish Immunol ; 93: 823-831, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422181

RESUMO

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.


Assuntos
Calreticulina/genética , Calreticulina/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Calreticulina/química , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Padrões Moleculares Associados a Patógenos/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
12.
Bioelectrochemistry ; 130: 107348, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31437810

RESUMO

A practical electrochemical biosensor with high sensitivity was developed for detecting organophosphorus (OP). Initially, Ce metal was introduced into an UiO-66-template to form Ce/UiO-66. Later, graphene oxide (GO), carbon black (CB) and multi-walled carbon nanotubes (MWCNTs) were separately added to Ce/UiO-66 to compare the effect of different carbon-based material types on the performance of the biosensor. Exclusively, Ce/UiO-66/MWCNTs with a Ce (7%) and MWCNT (30%) matrix was found to not only load more acetylcholinesterase (AChE) onto vacant sites but also increase electron transfer and decrease the number of diffusion pathways between the thiocholine and electrode surface. Moreover, the appropriate oxophilicity of Ce coupled with the high surface area and good conductivity of MWCNTs in the UiO-66 structure revealed a high affinity to acetylthiocholine chloride (ATCl) and possible catalysis of the hydrolysis of ATCl with a Michaelis-Menten constant of 0.258 mM. This biosensor, under optimal conditions, demonstrated a rapid and sensitive detection of paraoxon over a wide linear range of 0.01-150 nM, with a low detection limit of 0.004 nM. As a result, the AChE/Ce/UiO-66/MWCNTs/GCE biosensor can be employed in laboratory and field experiments to determine paraoxon levels.


Assuntos
Técnicas Biossensoriais/métodos , Cério/química , Estruturas Metalorgânicas/química , Paraoxon/análise , Praguicidas/análise , Acetilcolinesterase/química , Animais , Brassica/química , Electrophorus , Enzimas Imobilizadas/química , Proteínas de Peixes/química , Grafite/química , Modelos Moleculares , Nanotubos de Carbono/química , Spinacia oleracea/química
13.
Fish Shellfish Immunol ; 93: 597-611, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400511

RESUMO

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Lactococcus/fisiologia , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/imunologia , Alinhamento de Sequência/veterinária , Fator de Transcrição AP-1/química
14.
Fish Shellfish Immunol ; 93: 623-630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400512

RESUMO

Cathepsin S belong to the cathepsin L-like family of cysteine cathepsins. It is well known that Cathepsin S participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin S is less investigated. In the present study, a cathepsin S homologue (SsCTSS) from the teleost fish black rockfish (Sebastes schlegelii) were identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, were existed in the cathepsin. SsCTSS possesses a peptidase domain with three catalytically essential residues (Cys25, His162, and Asn183). Phylogenetic profiling indicated that SsCTSS are evolutionally close to the cathepsin S of other teleost fish. The expression of SsCTSS in immune-related tissues was upregulated in a time-dependent manner upon bacterial pathogen infection. Purified recombinant SsCTSS (rSsCTSS) exhibited apparent peptidase activity, which was remarkably declined in the presence of the cathepsin inhibitor E-64. rSsCTSS showed strong binding ability to LPS and PGN, the major constituents of the outer membranes of Gram-negative and Gram-positive bacteria, respectively. rSsCTSS also exhibited the capability of agglutination to different bacteria. The knockdown of SsCTSS attenuated the ability of host to eliminate pathogenic bacteria. Taken together, our results suggested that SsCTSS functions as cysteine protease which might be involved in the antibacterial immunity of black rockfish.


Assuntos
Catepsinas/genética , Catepsinas/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Catepsinas/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária
15.
Fish Shellfish Immunol ; 93: 683-693, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408729

RESUMO

Fish skin mucus is considered to act as the first line of defense against waterborne pathogens and to be potential source of novel antimicrobial components. Here we report the purification and characterization of a novel hepcidin type 2-like antimicrobial peptide (TpHAMP2) from the skin mucus of the pufferfish Takifugu pardalis. The purified TpHAMP2 comprised of 23 amino acids (AAs) with eight Cys residues that form four intramolecular disulfide bonds. The TpHAMP2 gene shared overall structural characteristics with all known hepcidins, which have a tripartite exon-intron gene organization and three structural signatures in the precursor protein. Phylogenetically, TpHAMP2 was classified as HAMP2 class in acanthopterygian fish. Interestingly, the AA sequence of TpHAMP2 did not contain a proprotein cleavage site (RXXR motif) that conserved in most hepcidins and showed a highly positive charged (RKR-) short N-terminus and Val18 and Gly22 residues, which are distinctive structures compared to other known active hepcidins. Recombinant TpHAMP2 identical to the native form exhibited a broad spectrum and potent antimicrobial activity against tested gram-positive and -negative bacteria. Expression of TpHAMP2 mRNA was predominant in the liver and was upregulated in the liver, the spleen, the intestine, and the skin of T. pardalis post immune challenge. Thus, our findings suggests that TpHAMP2 might be of importance in the framework of discovering the fish hepcidins, especially type 2s, and provide noteworthy insight into its gene structure and expression and in the innate immunity as well as the mucosal immunity in regard to hepcidins' evolutionary history in fish species.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Hepcidinas/genética , Hepcidinas/imunologia , Imunidade Inata/genética , Takifugu/genética , Takifugu/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Hepcidinas/química , Imunidade nas Mucosas/genética , Masculino , Filogenia , Alinhamento de Sequência/veterinária
16.
Food Chem ; 301: 125278, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387033

RESUMO

Softening is always a problem in fish preservation. This study was aimed to investigate the role of myofibrillar structural proteins degradation in fish softening. The changes of myofibrillar structural proteins, muscle ultrastructure, myofibril fragmentation, and shear force were studied. The results indicated that during the superchilled preservation of grass carp (Ctenopharyngodon idella), small (low-molecular-weight) myofibrillar structural proteins like desmin and troponin-T initiated textural deterioration, leading to Z-disk weakening and actin loosening. In contrast, giant (high-molecular-weight) myofibrillar structural proteins like titin and nebulin were degraded in more amount in the later storage, contributing to Z-disk and M-band disassembly and vague of light and dark regions (I and A bands). Compared to each other, desmin and titin played more important part in softening. All these changes were involved in the increase of muscle fibril segments and the sharp decrease of shear force.


Assuntos
Carpas/metabolismo , Produtos Pesqueiros , Proteínas de Peixes/química , Miofibrilas/química , Animais , Conectina/química , Conectina/metabolismo , Desmina/química , Desmina/metabolismo , Proteínas de Peixes/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Proteólise , Troponina T/química , Troponina T/metabolismo
17.
Fish Shellfish Immunol ; 93: 308-312, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352113

RESUMO

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Sequência de Aminoácidos , Animais , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fator 88 de Diferenciação Mieloide/química , Filogenia , Alinhamento de Sequência/veterinária
18.
Fish Shellfish Immunol ; 93: 449-462, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352119

RESUMO

Galectins are ß-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (-) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Galectinas/genética , Galectinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Galectinas/química , Perfilação da Expressão Gênica/veterinária , Bactérias Gram-Negativas/fisiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Bactérias Gram-Positivas/fisiologia , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária
19.
Fish Shellfish Immunol ; 93: 1056-1066, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31340170

RESUMO

In mammals, the multifunctional DExH/D-box helicases, DDX3 and DHX9, are nucleic acid sensors with a role in antiviral immunity; their role in innate immunity in fish is not yet understood. In the present study, full-length DDX3 and DHX9 coding sequences were identified in rainbow trout (Oncorhynchus mykiss). Bioinformatic analysis demonstrated both deduced proteins were similar to those of other species, with ~80% identity to other fish species and ~70-75% identity to mammals, and both protein sequences had conserved domains found amongst all species. Phylogenetic analysis revealed clustering of DDX3 and DHX9 with corresponding proteins from other fish. Cellular localization of overexpressed DDX3 and DHX9 was performed using GFP-tagged proteins, and endogenous DDX3 localization was measured using immunocytochemistry. In the rainbow trout gonadal cell line, RTG-2, DHX9 localized mostly to the nucleus, while DDX3 was found mainly in the cytoplasm. Tissue distribution from healthy juvenile rainbow trout revealed ubiquitous constitutive expression, highest levels of DDX3 expression were seen in the liver and DHX9 levels were fairly consistent among all tissues tested. Stimulation of RTG-2 cells revealed that DDX3 and DHX9 transcripts were both significantly upregulated by treatment with the dsRNA molecule, poly I:C. A pull-down assay suggested both proteins were able to bind dsRNA. In addition to their roles in RNA metabolism, the conserved common domains found between the rainbow trout proteins and other species having defined antiviral roles, combined with the ability for the proteins to bind to dsRNA, suggest these proteins may play an important role in fish innate antiviral immunity. Future studies on both DDX3 and DHX9 function will contribute to a better understanding of teleost immunity.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , RNA Helicases DEAD-box/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Poli I-C/farmacologia
20.
Fish Shellfish Immunol ; 93: 108-115, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326582

RESUMO

Protein arginine methylation is a prevalent posttranslational modification and protein arginine methyltransferases 6 (PRMT6) has been identified as a suppressor of TBK1/IRF3 in human and mammals. To explore the role of PRMT6 in teleost fish, PRMT6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp PRMT6 (bcPRMT6) transcription in host cells varies in response to different stimuli and bcPRMT6 migrates around 43 kDa in the immunoblot assay. Like its mammalian counterpart, bcPRMT6 has been identified to distribute majorly in the nucleus through the immunofluorescent staining assay. bcPRMT6 shows little interferon (IFN) promoter-inducing activity in the reporter assay and bcPRMT6 shows no antiviral activity against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV) in plaque assay. When co-expressed with bcPRMT6, the IFN promoter-inducing abilities of black carp TBK1 (bcTBK1) and IRF3/7 (bcIRF3/7) are fiercely attenuated. Accordingly, bcTBK1-mediated antiviral activity in EPC cells is obviously dampened by bcPRMT6. The interaction between bcPRMT6 and bcIRF3/7 has been identified by co-immunoprecipitation assay; however, no direct association between bcPRMT6 and bcTBK1 has been detected. Taken together, our data elucidates for the first time in teleost fish that PRMT6 suppresses TBK1-IRF3/7 signaling during host antiviral innate immune activation.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Filogenia , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/imunologia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Transdução de Sinais
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